CN114191561B - Application of ionizable lipid compound in nucleic acid drug delivery system - Google Patents
Application of ionizable lipid compound in nucleic acid drug delivery system Download PDFInfo
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
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- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
- A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
- A61K48/0033—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being non-polymeric
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
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- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
Abstract
The invention provides an application of an ionizable lipid compound in a nucleic acid drug delivery system. Most of the existing nucleic acid drug carriers have the problems of low in-vivo delivery efficiency or liver accumulated toxic and side effects and the like, and in order to solve the problems, the invention provides a novel drug delivery system. The nucleic acid drug delivery system of the present invention is suitable for various administration modes, has low toxic and side effects on liver and high nucleic acid delivery efficiency, and can efficiently deliver nucleic acid drug molecules into spleen with high specificity and effectively translate the nucleic acid drug molecules into target molecules.
Description
Technical Field
The invention belongs to the technical field of nucleic acid drug delivery, and particularly relates to an application of an ionizable lipid compound in a nucleic acid drug delivery system.
Background
Active molecules of nucleic acid drugs, including messenger rna (mrna), small interfering rna (sirna), antisense oligonucleotides (ASO) or plasmid dna (pdna), have great potential for use in vaccines and gene therapy in vitro or in vivo. How to successfully deliver nucleic acid drug molecules to cells in vivo and express them with high efficiency is one of the technologies which are urgently needed at present. Most of the existing nucleic acid drug carriers have the problems that the nucleic acid drug carriers are easy to accumulate at the liver part after entering the body, increase the metabolic burden of the liver and cause obvious toxic and side effects. Or toxic and side effects due to increased dosage caused by low delivery efficiency of the delivery system. Therefore, the development of a delivery system with low toxic and side effects and high nucleic acid delivery efficiency is of great practical significance.
Disclosure of Invention
The invention aims to provide application of an ionizable lipid compound in a nucleic acid drug delivery system, wherein the nucleic acid drug delivery system can deliver nucleic acid drug molecules into a body efficiently and realize efficient expression of nucleic acid, and the nucleic acid expression level in liver is low, so that toxicity can be reduced.
In order to solve the technical problems, the invention adopts the following technical scheme:
use of an ionizable lipid compound in a nucleic acid drug delivery system capable of delivering a nucleic acid drug molecule to the spleen and/or lung, said ionizable lipid compound being one or more of compounds of formula (i), formula (ii), and formula (iii):
wherein the content of the first and second substances,
r is-OC (═ O) -or-C (═ O) O-.
In the present invention, the structure of R is defined in the direction from one end of the chain to the other end, i.e., when R is — OC (═ O) -, O in the ester group is adjacent to N, and when R is — C (═ O) O-, C in the ester group is adjacent to N.
R 1 Is hydrogen, methyl, ethyl or isopropyl.
m is an integer between 1 and 10, for example m is 1,2, 3, 4, 5, 6, 7, 8, 9 or 10.
n is an integer of 1 to 3, for example, n is 1,2 or 3.
f is an integer of 1 to 5, for example f is 1,2, 3, 4 or 5.
x is an integer between 1 and 8, for example x is 1,2, 3, 4, 5, 6, 7 or 8.
y is an integer between 1 and 9, for example y is 1,2, 3, 4, 5, 6, 7, 8 or 9.
R 2 And R 3 Independently hydrogen, methyl, ethyl or isopropyl.
p is an integer of 1 to 5, for example, p is 1,2, 3, 4 or 5.
q is an integer between 1 and 3, for example q is 1,2 or 3.
Preferably, said R is-C (═ O) O-.
Preferably, R is 1 Is hydrogen.
Preferably, R is 2 And said R 3 One of which is hydrogen and the other is methyl, ethyl or isopropyl.
Further preferably, R is 2 And said R 3 One of which is hydrogen and the other is methyl.
Preferably, m is an integer between 3 and 8, and more preferably an integer between 4 and 6.
Preferably, f is an integer between 1 and 4, and is further preferably 2 or 3.
Preferably, x is an integer between 2 and 5, and more preferably an integer between 2 and 4.
Preferably, y is an integer between 3 and 9, and more preferably an integer between 5 and 9.
Preferably, said R is-C (═ O) O-, said R is 1 Is hydrogen, said n is 2, said R 2 And said R 3 One is hydrogen and the other is methyl, and p is 5.
According to some embodiments, in formula (I), R 1 Is hydrogen, m is an integer of 4 to 6, n is 2, and f is an integer of 1 to 3.
According to some embodiments, in formula (II), x is 3 and y is an integer between 6 and 9.
According to some embodiments, in formula (III), R 2 Is methyl, R 3 P is hydrogen, p is 5, and q is an integer between 1 and 3.
According to some preferred embodiments, the ionizable lipid compound is one or more of the compounds represented by the following structural formula:
preferably, the nucleic acid drug molecule is one or more of mRNA, siRNA, ASO or pDNA (plasmid DNA).
Further preferably, the mass ratio of the nucleic acid drug molecule to the nucleic acid drug delivery system is 1 (5-50), further preferably 1 (5-40), further preferably 1 (5-30), and further preferably 1 (5-20).
Further preferably, the delivery system forms a nano-lipid particle with the delivered nucleic acid drug, and the average size of the nano-lipid particle is 50nm to 200 nm.
More preferably, the polydispersity index of the nano-lipid particles is less than or equal to 0.4.
Further, the average size of the nano-lipid particles is 50 nm-150 nm.
Further, the polydispersity index of the nano-lipid particles is less than or equal to 0.3.
Preferably, the ionizable lipid compound is optionally modified with a targeting substance, which is one or more of folic acid, a single chain antibody, or a targeting polypeptide.
Preferably, the nucleic acid drug delivery system further comprises an auxiliary molecule selectively carrying a targeted substance modification, and the feeding molar ratio of the ionizable lipid compound to the auxiliary molecule is (0.1-1): (0.1-1), more preferably (0.5-1): (0.5 to 1).
The helper molecules include helper lipid or lipoid molecules commonly used in the art.
Preferably, the auxiliary molecules include, but are not limited to, one or more of cholesterol, calcipotriol, stigmasterol, β -sitosterol, betulin, lupeol, ursolic acid, oleanolic acid, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, 1-stearoyl-2-oleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, (1, 2-dioleoxypropyl) trimethylammonium chloride, didecyldimethylammonium bromide, 1, 2-dimyristoyl-sn-glycero-3-ethylphosphonium chloride, dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol 5000, distearoylphosphatidylethanolamine-polyethylene glycol 2000.
Preferably, the targeting substance is one or more of folic acid, a single chain antibody or a targeting polypeptide.
In the present invention, the modification of the ionizable lipid compound or accessory molecule with a targeting substance can be performed using methods conventional in the art.
According to some embodiments, Mal-PEG2000-DSPE and polypeptide can be dissolved in ultrapure water, stirred to react for 48 hours, dialyzed and concentrated to obtain polypeptide-PEG 2000-DSPE, and the targeting auxiliary molecule can be obtained.
Further, the ionizable lipid molecule: cholesterol: DOPE: dissolving polypeptide-PEG 2000-DSPE in anhydrous ethanol, and mixing with DNA or RNA to form polypeptide modified liposome.
Preferably, the nucleic acid drug delivery system is an injection.
Preferably, the nucleic acid drug delivery system further comprises an additive, wherein the additive comprises a stabilizer and/or a diluent.
Preferably, the additive is added in an amount of 1 to 20% by weight of the total mass of the injection.
The additives may be those commonly used in the art.
Preferably, the stabilizer includes, but is not limited to, sucrose or trehalose.
Preferably, the diluent includes, but is not limited to, buffers commonly used in the art, including, but not limited to, one or more of phosphate buffer, acetate buffer, tris hydrochloride buffer.
Preferably, the nucleic acid drug delivery system is administered by local intramuscular, subcutaneous, endothelial, intratumoral injection or perfusion, or by intravenous injection.
The nucleic acid drug delivery system can deliver nucleic acid drug molecules such as mRNA, siRNA or pDNA into a body through various administration modes such as local muscle, subcutaneous, endothelial, intratumoral and perfusion, and systemic administration modes such as intravenous injection, and can even target spleen/lung, effectively express therapeutic protein drugs or antigens in cells in the body, and play a role in preventing or treating diseases. The nucleic acid drug delivery system of the present invention can significantly reduce the liver accumulation effect of liposomes, and by selecting an appropriate administration mode, nucleic acids can be efficiently delivered into the spleen or lung and efficiently translated into target molecules, and thus the nucleic acid drug delivery system of the present invention has the potential to be suitable for delivering nucleic acid drugs into the body to function as a prophylactic or therapeutic vaccine.
Compared with the prior art, the invention has the following advantages:
the nucleic acid drug delivery system can efficiently deliver nucleic acid drug molecules into spleen and/or lung and effectively translate the nucleic acid drug molecules into target molecules, can reduce the side effect of liposome accumulation in liver, and has important significance for the development and application of nucleic acid drugs.
Drawings
FIG. 1 is a hydrogen spectrum of Compound 1-1;
FIG. 2 is a hydrogen spectrum of 2- (bis (2-aminoethyl) amino) ethan-1-ol);
FIG. 3 is a hydrogen spectrum of Compound 1;
FIG. 4 is a mass spectrum of Compound 1;
FIG. 5 is a hydrogen spectrum of Compound 2-1;
FIG. 6 is a hydrogen spectrum of Compound 2;
FIG. 7 is a mass spectrum of Compound 2;
FIG. 8 is a hydrogen spectrum of Compound 3-1;
FIG. 9 is a hydrogen spectrum of 1, 3-diamino-2-propanol;
FIG. 10 is a hydrogen spectrum of Compound 3;
FIG. 11 is a mass spectrum of Compound 3;
FIG. 12 is a graph showing a particle size distribution of Lipid-1, Lipid-2 and Lipid-3 in example 4;
FIG. 13 shows the fluorescence expression at different time points after intramuscular injection of Lipid-1 in mice;
FIG. 14 shows the fluorescence expression of each organ of mice after intravenous injection and intramuscular injection of Lipid-16 h;
FIG. 15 shows the fluorescence expression at different time points after intramuscular injection of Lipid-2 in mice;
FIG. 16 shows the fluorescence expression of the organs of mice after intravenous injection and intramuscular injection of Lipid-26 h;
FIG. 17 shows the fluorescence expression at different time points after intramuscular injection of Lipid-3 in mice;
FIG. 18 shows the fluorescence expression of each organ of mice after intravenous injection and intramuscular injection of Lipid-36 h;
FIG. 19 shows the in vivo fluorescence expression of mice injected with Lipid-1, Lipid-2, Lipid-36 h intravenously in comparison with SM-102.
Detailed Description
The present invention will be further described with reference to the following examples. However, the present invention is not limited to the following examples. The implementation conditions adopted in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions not mentioned are conventional conditions in the industry. The technical features of the embodiments of the present invention may be combined with each other as long as they do not conflict with each other.
In order to effectively package and deliver exogenous nucleic acid drugs into specific organs and tissues in vivo and efficiently express the molecules, the inventors have conducted a great deal of research on major lipid compounds of nucleic acid drug delivery systems, and finally invented a novel nucleic acid drug delivery system capable of delivering nucleic acid drug molecules to specific organs or tissues and achieving efficient expression of nucleic acids. Compared with the MC3 system commonly used in the field, the expression level of the target nucleic acid drug molecule is similar to or higher, and the liver accumulation degree is obviously reduced.
According to the present invention, the drug delivery system is capable of delivering nucleic acid drug molecules to the spleen and/or lung with low accumulation in the liver, and comprises an ionizable lipid compound, wherein the ionizable lipid compound is one or more of compounds represented by general formula (i), general formula (ii), and general formula (iii):
wherein the content of the first and second substances,
r is-OC (═ O) -or-C (═ O) O-; r 1 Is hydrogen, methyl, ethyl or isopropyl; m is an integer between 1 and 10; n is an integer between 1 and 3; f is an integer between 1 and 5; x is an integer between 1 and 8; y is an integer between 1 and 9; r 2 And R 3 Independently hydrogen, methyl, ethyl or isopropyl; p is an integer between 1 and 5; q is an integer of 1 to 3.
In the invention, the nucleic acid drug delivery system is a nano lipid particle.
In vivo experiments show that mRNA, siRNA or pDNA nucleic acid molecules can be mostly delivered to spleen or lung by various administration modes such as local muscle, subcutaneous, endothelial, intratumoral and perfusion, or by systemic administration, and the distribution ratio of the mRNA, the siRNA or the pDNA nucleic acid molecules in the liver is obviously reduced. The nucleic acid drug delivery system of the present invention is advantageous in that spleen and/or lung can be selectively targeted even without a targeting group attached, with little or negligible distribution in the liver, resulting in lower hepatotoxicity. This is the most significant feature of the nucleic acid drug delivery system of the present invention compared to most existing nucleic acid drug delivery systems, and is an advantageous attribute beyond that of a general delivery vehicle that can prevent the degradation of the loaded nucleic acid drug molecules (especially mRNA and siRNA) in vivo by nucleases.
The technical solution and the advantages of the present invention are further illustrated by the following specific examples.
The experimental methods in the following examples are all conventional methods unless otherwise specified; the experimental materials used, unless otherwise specified, were purchased from conventional biochemical manufacturers.
In the following examples, Lipid-1 refers only to the nano Lipid particle formed using the compound 1 prepared in example 1 as an ionizable Lipid compound with other components, Lipid-2 refers only to the nano Lipid particle formed using the compound 2 prepared in example 2 as an ionizable Lipid compound with other components, and Lipid-3 refers only to the nano Lipid particle formed using the compound 3 prepared in example 3 as an ionizable Lipid compound with other components, without limitation on mRNA encapsulated therein.
SM102 as a control, the mRNA formed nano-lipid particles were encapsulated using the existing liposome SM-102.
Lipo2000 served as a control, and the existing liposome Lipo2000 was used to encapsulate the nano-lipid particles formed by the plasmids carrying the green fluorescent GPF tags.
Example 1
Synthetic route to compound 1:
step 1: synthesis of Compound 1-1:
linalool (0.267g, 1mmol) and triethylamine (0.133g, 1.3mmol) were added to a reaction flask in an ice-water bath, dichloromethane (6mL) was added, acryloyl chloride (0.11g, 1.2mmol) was dissolved in dichloromethane (2.2mL), slowly added dropwise to the reaction flask, the reaction was continued for 10 minutes, the reaction was maintained below 10 ℃, finally the ice bath was removed, and the reaction was allowed to react at room temperature for 2 hours. Washing with saturated brine gave a crude product which was purified by chromatography (silica gel column, eluent petroleum ether containing 0.5% EA (volume%) and the pure product was evaporated to give the compound 1-1 (9Z, 12Z) -octadecadienyl 2-enepropionate) (0.173g, yield: 50%) as a pale yellow oil, the hydrogen spectrum of compound 1-1 being shown in fig. 1.
1H NMR(400MHz,CDCl 3 )δ:6.41(dd,J＝17.3,1.5Hz,1H),6.13(dd,J＝17.3,10.4Hz,1H),5.82(dd,J＝10.4,1.5Hz,1H),5.47-5.26(m,4H),4.16(t,J＝6.7Hz,2H),2.78(t,J＝6.5Hz,2H),2.06(dd,J＝13.6,6.7Hz,4H),1.75-1.60(m,2H),1.39-1.17(m,16H),0.88(dt,J＝10.4,5.3Hz,3H).
Step 2: synthesis of Compound 1:
2- (bis (2-aminoethyl) amino) ethan-1-ol) (0.0735g, 0.50mmol, hydrogen spectrum see FIG. 2) and (9Z, 12Z) -octadecadienoic acid 2-ol (0.64g, 2mmol) were added to a reaction flask and reacted at 80 ℃ for 48 hours. After the reaction was cooled to room temperature, the solvent was removed in vacuo to give a crude product, which was purified by chromatography (silica gel column, eluent 0.5% methanol (volume%) in dichloromethane, and the pure product was evaporated to give compound 1 as a yellow oil (25.7mg, yield: 3.6%). the hydrogen spectrum of the compound is shown in FIG. 3, and the mass spectrum is shown in FIG. 4.
1H NMR(400MHz,CDCl 3 )δ5.45-5.30(m,16H),4.07(t,J＝6.8Hz,8H),3.52(s,1H),2.80(dd,J＝12.5,6.4Hz,16H),2.63(s,4H),2.54(s,3H),2.48(t,J＝7.2Hz,8H),2.07(q,J＝6.7Hz,16H),1.62(dd,J＝13.4,6.6Hz,8H),1.42-1.25(m,68H),0.91(t,J＝6.8Hz,12H),0.91(t,J＝6.8Hz,1H)。
Example 2
Synthetic route to Compound 2
Step 1: synthesis of Compound 2-1
6-Bromohexanoic acid (1.0g, 5.13mmol) and undecanol (1.77g, 10.25mmol) were dissolved in dichloromethane (60mL), and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC hydrochloride, 0.98g, 5.13mmol) and DMAP (0.125g, 1.03mmol) were added. The mixture was stirred at ambient temperature for 18 hours. After the reaction was complete, it was diluted with DCM (200mL) and saturated NaHCO 3 (100mL) and brine (100 mL). The organic layers were combined with anhydrous Na 2 SO 4 Drying and removal of the solvent in vacuo afforded the crude product which was purified by chromatography (silica gel column, eluent petroleum ether containing 0.5% EA (volume%) and the pure product was evaporated to afford 2-1 (undecyl 6-bromohexanoate) as a pale yellow oil (0.69g, 38.6% yield). The hydrogen spectrum of compound 2-1 is shown in FIG. 5.
1H NMR(400MHz,CDCl 3 )δ:4.10(t,J＝6.6Hz,2H),3.45(t,J＝6.7Hz,2H),2.36(t,J＝7.3Hz,2H),1.97-1.88(m,2H),1.68(tt,J＝14.5,7.3Hz,4H),1.53(dd,J＝15.1,7.9Hz,2H),1.33(d,J＝16.9Hz,16H),0.92(t,J＝6.5Hz,3H).
Step 2: synthesis of Compound 2
2- (bis (2-aminoethyl) amino) ethan-1-ol) (0.044g, 0.30 mmol)) And undecyl 6-bromohexanoate (0.417g, 1.20mmol) in THF/CH 3 CN (1:1, 6mL), followed by additional DIPEA (0.155g, 1.20 mmol). The reaction was stirred at 63 ℃ for 72h, cooled to room temperature and the solvent removed in vacuo. The crude product was extracted with ethyl acetate and saturated NaHCO 3 Extracting, combining organic layers and using anhydrous Na 2 SO 4 Drying and removal of the solvent in vacuo afforded the crude product which was purified by chromatography (silica gel column, eluent dichloromethane containing 1-2% methanol by volume) and the pure product was evaporated to afford compound 2 as a yellow oil (14.64mg, 4% yield). The hydrogen spectrum of compound 2 is shown in FIG. 6, and the mass spectrum is shown in FIG. 7.
1H NMR(400MHz,CDCl 3 )δ4.10-3.98(m,8H),3.63(d,J＝16.5Hz,2H),3.46(s,1H),3.22(d,J＝45.3Hz,3H),3.05(d,J＝4.0Hz,2H),3.01-2.73(m,9H),2.66(d,J＝14.3Hz,2H),2.33(dd,J＝16.6,7.4Hz,8H),1.98(s,2H),1.78(s,2H),1.64(dt,J＝23.5,7.0Hz,20H),1.41-1.20(m,74H),0.88(t,J＝6.7Hz,12H)。
Example 3
Synthetic route to compound 3
Step 1: synthesis of Compound 3-1
8-Bromocaprylic acid (1.139g, 5.13mmol) and 3, 7-dimethyloct-6-en-1-ol (citronellol, 1.599g, 10.25mmol) were dissolved in dichloromethane (60mL) and, after sufficient dissolution, EDC hydrochloride (0.98g, 5.13mmol) and DMAP (0.125g, 1.03mmol) were added. The mixture was stirred at ambient temperature for 18 hours. After the reaction was complete, it was diluted with DCM (200mL) and saturated NaHCO 3 (100mL) and brine (100 mL). The organic layers were combined with anhydrous Na 2 SO 4 Drying and removal of the solvent in vacuo afforded a crude product which was purified by chromatography (silica gel column, eluent petroleum ether containing 0.5% EA (volume%) and the pure product was evaporated to afford compound 3-1 (3, 7-dimethyloct-6-enyl 6-bromohexanoate) (0.648g, 35%) as a pale yellow oil, compound 3-1, a hydrogen spectrum as shown in figure 8.
1H NMR(400MHz,CDCl 3 )δ:5.09(s,1H),4.18-4.01(m,2H),3.40(t,J＝6.8Hz,2H),2.29(t,J＝7.4Hz,2H),1.98(s,2H),1.84(dd,J＝14.3,7.0Hz,2H),1.70-1.60(m,9H),1.38(d,J＝37.7Hz,9H),0.89(t,J＝12.9Hz,4H).
Step 2: synthesis of Compound 3
1, 3-diamino-2-propanol (0.027g, 0.30mmol, hydrogen spectrum shown in FIG. 9) and 3, 7-dimethyloct-6-enyl 6-bromohexanoate (0.398g, 1.2mmol) were added to a reaction flask and dissolved in THF/CH 3 CN (1:1, 6mL), followed by additional DIPEA (0.155g, 1.20 mmol). The reaction was stirred at 63 ℃ for 72h, cooled to room temperature and the solvent removed in vacuo. The crude product was extracted with ethyl acetate and saturated NaHCO 3 Extracting, combining organic layers and using anhydrous Na 2 SO 4 Drying and removal of the solvent in vacuo afforded the crude product which was purified by chromatography (silica gel column, eluent dichloromethane containing 1% methanol (vol.%), and the pure product was evaporated to afford compound 3 as a pale yellow oil (11.63mg, 3.2% yield). The hydrogen spectrum of compound 3 is shown in FIG. 10, and the mass spectrum is shown in FIG. 11.
1H NMR(400MHz,CDCl 3 )δ5.30(s,1H),5.08(t,J＝6.4Hz,4H),4.10(h,J＝10.9Hz,8H),3.67(s,1H),2.46(s,10H),2.28(t,J＝7.3Hz,8H),1.97(d,J＝9.2Hz,8H),1.74-1.51(m,41H),1.49-1.37(m,12H),1.37-1.16(m,33H),0.91(d,J＝5.9Hz,12H)。
Example 4: preparation of LNP-mRNA nano-lipid particles, measurement of particle size and potential:
dissolving and mixing the compound 1, the compound 2 and the compound 3 prepared in the examples 1 to 3 with DSPC, DMG-PEG2000 and cholesterol respectively according to a molar ratio of 50:10:1.5:38.5 by using absolute ethyl alcohol as a solvent to obtain a liposome raw material solution, controlling the sum of the concentrations of the components to be 50mM, completely dissolving and uniformly mixing, and then standing at-20 ℃ for storage.
The mRNA was dissolved in 25mM sodium acetate buffer solution at a pH of about 5.2 to prepare a nucleic acid preparation having a final concentration of about 0.1 mg/mL.
The prepared liposome raw material solution and the nucleic acid preparation are mixed uniformly rapidly through a Nano Assembly r micro-fluidic system or a vortex method under the conditions that the volume ratio of two phases is about 4:1 and the total speed of the two-phase solution is 12mL/min to form uniform and stable Nano liposome particles, and then the environment of the Nano liposome particles is rapidly changed from pH 5.2 to 7.0-7.4. Specifically, diluting the liposome particles by using PBS buffer solution with pH of 7.2 or sodium acetate buffer solution with pH of 7.4 for 20 times of volume, concentrating by using an ultrafiltration tube with 10KD, wherein the rotation speed bj of a centrifuge is not more than the maximum rotation speed limit of the ultrafiltration tube, after 2-3 times of liquid change, the pH of the solution environment of the nano liposome particles is about 7.2-7.4, concentrating the nano liposome particles to the final concentration of about 200mM, and placing the nano liposome particles in the environment at 4 ℃ for later use, wherein the nano liposome particles are respectively marked as Lipid-1, Lipid-2 and Lipid-3.
The particle size of the nanoliposome particles, PDI, was measured using Zetasizer Nano ZS (Malvern, Worcestershire, UK). The particle size was measured by diluting the nanoliposome particle solution 50 times with 1 XPBS, the Zeta potential was measured by diluting the nanoliposome particles into 15mM PBS, and the encapsulation efficiency was measured on a Modulus microporous multifunctional detector using Quant-It RiboGreenRNA quantitative detection kit. The results of measurement of particle size, PDI, encapsulation efficiency, and potential are shown in table 1 and fig. 12.
TABLE 1 physical parameters of representative LNP-mRNA nanoliposome particles
The result shows that the average particle size of the Lipid-1, Lipid-2 and Lipid-3 is 150nm, the particle size distribution is uniform, the encapsulation efficiency is more than 90 percent, and the potential is-20.8 mV to-10.2 mV.
Example 5: in vivo transfection experiments in animals with nano-lipid particles:
the preparation method of example 4 was performed using intramuscular injection of nano-liposome particles to prepare nano-liposome particles, wherein the mRNA was mRNA expressing Luciferase protein, the amount of the mRNA was 60 μ g, the total amount of ionizable liposome compound, DSPC, DMG-PEG2000, and cholesterol was 600 μ g, 200 μ L of neutral PBS buffer was used to rapidly switch liposome environment, and the cells were rapidly injected into the medial muscle of hind limb of female Babl/c mice for 6-8 weeks, and the left and right hind limbs were controlled to be injected with 30 μ g of mRNA, respectively, or injected intravenously into female Babl/c mice for 6-8 weeks, and 50 μ g of mRNA was controlled to be intravenously injected.
And (3) carrying out intramuscular injection of Lipid-1, Lipid-2 and Lipid-3 on the leg of the mouse, carrying out fluorescence expression of mRNA at different time points in 4h, 24h, 48h and 72h in the mouse, and integrating fluorescence intensity inside a circle to obtain a fluorescence intensity numerical value for quantitatively representing the expression quantity of luciferase and drawing a column diagram. The fluorescence intensity in the figure is closely related to the delivery efficiency of the liposome, and the stronger the fluorescence, the higher the delivery efficiency of the liposome is represented.
The fluorescence expression at different time points after intramuscular injection of Lipid-1 in mice is shown in FIG. 13. The mean fluorescence expression amount after 4 hours after injecting Lipid-1 intramuscularly to the legs of mice was 3.5X 10 7 Since the amount of fluorescence of Lipid-1 in the liver region is significantly reduced as compared with the reference standard MC3 and SM-102 of Moderna in this field, the metabolic burden that the liver needs to bear is low. And the fluorescent expression can last for 72h, which shows that the liposome has good protection effect on mRNA.
The fluorescence expression of each organ after intravenous injection and intramuscular injection of Lipid-16 h in mice is shown in FIG. 14. The results showed that the fluorescence expression in each organ of the mouse was approximately 56% in spleen, 34% in liver and 10% in lung, respectively, after intravenous injection of the liposome of Lipid-1 (IV); after intramuscular Injection (IM), the fluorescence expression levels in each organ of the mouse were approximately 60% in the spleen, 30% in the liver and 10% in the lung, respectively.
The fluorescence expression at different time points after intramuscular injection of Lipid-2 in mice is shown in FIG. 15. The mean fluorescence expression amount after 4 hours after intramuscular injection of Lipid-2 into the legs of mice was 4.0X 10 7 And the fluorescence expression amount of the muscle tissue is still as high as 1.0 multiplied by 10 after 24h 6 . The distribution of fluorescence at the liver site was significantly reduced compared to MC3 and SM 102.
The fluorescence expression of each organ after intravenous injection of Lipid-26 h in mice is shown in FIG. 16. As a result, the fluorescence expression levels in the respective organs of the mouse after intravenous injection of the liposome of Lipid-2 (IV) were about 92% in the liver and 8% in the spleen, respectively.
The fluorescence expression at different time points after intramuscular injection of Lipid-3 in mice is shown in FIG. 17. The mean fluorescence expression amount after 4 hours after injecting Lipid-3 intramuscularly to the legs of mice was 1.0X 10 6 And fluorescence expression can still be maintained at approximately this level after a duration of 48 h. The distribution of fluorescence at the liver site was significantly reduced compared to MC3 and SM 102.
The fluorescence expression of each organ of mice after intravenous injection of Lipid-36 h is shown in FIG. 18. The results showed that the fluorescence expression in each organ of the mice was approximately 88% in spleen and 12% in liver after Intravenous (IV) Lipid-3 liposome injection, while the fluorescence expression in spleen after intramuscular injection was a little lower than that of the mice after intravenous injection.
When the liposome containing Lipid-1, Lipid-2, Lipid-3 and SM102 was injected into mice through tail vein, a distinct fluorescence distribution appeared in each mouse after 6h, as shown in FIG. 19, the fluorescence of SM-102 and Lipid-2 liposomes was mainly distributed in the liver region, and the fluorescence of Lipid-1 and Lipid-3 liposomes was mainly distributed in the spleen region, which is consistent with the fluorescence distribution of organs dissected from mice. The Lipid-1 and Lipid-3 liposomes have obvious spleen targeting effect, are suitable for local administration by intramuscular injection or systemic administration by intravenous injection, have low hepatotoxicity, and the Lipid-2 is more suitable for an intramuscular injection administration mode.
The present invention has been described in detail in order to enable those skilled in the art to understand the invention and to practice it, and it is not intended to limit the scope of the invention, and all equivalent changes and modifications made according to the spirit of the present invention should be covered by the present invention.
Claims (8)
1. Use of an ionizable lipid compound for the preparation of a nucleic acid drug delivery system, wherein: the nucleic acid drug delivery system is capable of delivering nucleic acid drug molecules to the spleen and/or lung, and the ionizable lipid compound is a compound shown in the following structural formula:
2. Use of an ionizable lipid compound according to claim 1 for the preparation of a nucleic acid drug delivery system, wherein said nucleic acid drug molecule is one or more of mRNA, siRNA, ASO or pDNA;
and/or the mass ratio of the nucleic acid drug molecule to the nucleic acid drug delivery system is 1 (5-50).
3. Use of an ionizable lipid compound according to claim 1 for the preparation of a nucleic acid drug delivery system, wherein said nucleic acid drug delivery system is a nano-lipid particle having an average size of 50nm to 200 nm.
4. Use of an ionizable lipid compound according to claim 1 for the preparation of a nucleic acid drug delivery system, wherein said ionizable lipid compound is optionally modified with a targeting substance, said targeting substance being one or more of folic acid, a single chain antibody or a targeting polypeptide.
5. Use of an ionizable lipid compound according to claim 1 for the preparation of a nucleic acid drug delivery system, further comprising an auxiliary molecule optionally modified with a targeting substance, wherein said ionizable lipid compound and said auxiliary molecule are present in a molar ratio of (0.1-1): (0.1 to 1); the auxiliary molecules comprise one or more of cholesterol, calcipotriol, stigmasterol, beta-sitosterol, betulin, lupeol, ursolic acid, oleanolic acid, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, 1-stearoyl-2-oleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, (1, 2-dioleoxypropyl) trimethylammonium chloride, didecyl dimethyl ammonium bromide, 1, 2-dimyristoyl-sn-glycerol-3-ethylphosphonic acid choline, dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol 5000 and distearoylphosphatidylethanolamine-polyethylene glycol 2000; the target substance is one or more of folic acid, a single-chain antibody or a target polypeptide.
6. A nucleic acid drug delivery system comprising an ionizable lipid compound that is:
The nucleic acid drug delivery system is characterized by being an injection, and further comprising an additive, wherein the additive comprises a stabilizer and/or a diluent, and the additive amount of the additive is 1% -20% of the total mass of the injection.
7. The nucleic acid drug delivery system of claim 6, wherein the stabilizer comprises sucrose or trehalose; the diluent comprises one or more of phosphate buffer solution, acetate buffer solution, citrate and tris hydrochloride buffer solution.
8. The nucleic acid drug delivery system of claim 6, wherein said nucleic acid drug delivery system is administered by local intramuscular, subcutaneous, endothelial, intratumoral injection or perfusion, or by intravenous injection.
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| CN114191561B (en) * | 2021-12-15 | 2022-08-02 | 武汉滨会生物科技股份有限公司 | Application of ionizable lipid compound in nucleic acid drug delivery system |
| CN114262275B (en) * | 2021-12-15 | 2023-09-29 | 华中师范大学 | High-efficiency low-toxicity DNA and RNA lipid delivery carrier |
| CN114716355B (en) * | 2022-04-02 | 2023-09-05 | 华南理工大学 | Lipid compound, composition containing lipid compound and application of lipid compound |
| CN115957329A (en) * | 2022-10-25 | 2023-04-14 | 中南大学 | Use of ionizable lipid compounds in the preparation of nucleic acid drug delivery systems |
| CN115925563B (en) * | 2023-02-28 | 2023-05-16 | 清华大学 | Lipid molecule for targeted pulmonary delivery of nucleic acid, and preparation method and application thereof |
| CN116199646B (en) * | 2023-03-07 | 2024-01-26 | 山东大学 | Tris-based ionizable lipid, and preparation method and application thereof |
| CN116284006B (en) * | 2023-05-10 | 2023-08-25 | 北京因诺惟康医药科技有限公司 | Ionizable lipid compounds, lipid carriers comprising same and uses thereof |
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| CN114191561B (en) * | 2021-12-15 | 2022-08-02 | 武汉滨会生物科技股份有限公司 | Application of ionizable lipid compound in nucleic acid drug delivery system |
| CN114262275B (en) * | 2021-12-15 | 2023-09-29 | 华中师范大学 | High-efficiency low-toxicity DNA and RNA lipid delivery carrier |
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| EP3315125A1 (en) * | 2016-10-31 | 2018-05-02 | Silence Therapeutics (London) Ltd | Lipid nanoparticle formulation |
| CN111315359A (en) * | 2017-08-31 | 2020-06-19 | 摩登纳特斯有限公司 | Method for preparing lipid nanoparticles |
| CN111954542A (en) * | 2018-02-01 | 2020-11-17 | 塔夫茨大学信托人 | Lipid-like nanocomposite and use thereof |
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